Mechanisms of Endocytosis in Adrenal Chromaffin Cells.

肾上腺染色质细胞内吞作用的机制应激是对恶劣环境的生理反应。压力导致肾上腺的染色质细胞增加释放化学递质，包括肾上腺素，进入血液。这种释放是通过含有被称为分泌颗粒的递质包与细胞内部表面的融合来实现的，导致它们的内容物从细胞中排出。在正常情况下，嗜铬细胞释放低水平的肾上腺素，通过引导血液流向内部器官，使眼睛聚焦在附近的物体上，增加胰腺的胰岛素释放，使机体处于“繁殖和喂养”的能量储存状态。有压力时，染色质细胞会释放高水平的肾上腺素，导致“战斗或逃跑”反应。在这种状态下，升高的肾上腺素将血液流向骨骼肌，使他们的目光集中在远处的物体上，并提高血糖水平。此外，应激激活导致染色质细胞释放镇痛化合物，称为脑啡肽。这些信号分子一起使生物体进入一种逃避或防御的状态。因此，染色质细胞中受调节的递质释放在决定生物体的生理和代谢状态中起着至关重要的作用。本实验室的总体目标是提供对染色质细胞作为“繁殖和喂养”以及“战斗或逃跑”状态的主要贡献者的基本理解。这一建议侧重于染色质细胞在基础和应激激活下释放递质的机制。传递素的持续释放需要含有传递素的分泌颗粒的再循环。如果不循环利用，电池很快就会耗尽它的电量。从细胞表面提取特殊的颗粒成分，并重新填充传递分子以供重复使用。假定这种再循环在所有条件下都以同样的机制发生。然而，最近的研究表明，在基础激活和应力激活下，回收过程存在差异。本提案概述了一系列旨在测试特征事件的实验，这些事件描述了小鼠肾上腺染色质细胞在基础和应激激活条件下的循环机制。它提供了实验，将测试颗粒融合和崩溃的程度，在递质释放和颗粒和细胞表面组分混合的程度，从而确定在再生有活力的分泌颗粒之前所需的回收过程的复杂性。它还将确定参与循环机制的关键分子的作用，从而确定分泌颗粒再生的潜在调节点。这些实验中使用的技术包括单个细胞表面积的电生理测量、释放的递质量的电化学测定、细胞表面成分的定量荧光成像和循环机制的分子破坏。因此，将提供肾上腺染色质细胞在正常的“繁殖和喂养”以及“战斗或逃跑”应激激活状态下持续释放肾上腺素和脑啡肽的机制的基本描述。

Mechanisms of Endocytosis in Adrenal Chromaffin CellsCorey B. SmithCase Western Reserve UniversityStress is a physiological response to a hostile environment. Stress causes chromaffin cells of the adrenal gland to increase their release of chemical transmitters, including adrenaline, into the blood stream. This release is achieved by fusion of transmitter containing packets, called secretory granules, with the inside cell surface, causing their contents to be expelled from the cell. Under normal conditions, chromaffin cells release low levels of adrenaline and set the organism in a 'breed and feed' state of energy storage by directing blood flow to the internal organs, focusing the eye on nearby objects and increasing insulin release from the pancreas. With stress, chromaffin cells release elevated levels of adrenaline, contributing to the 'fight or flight' response. In this state, the elevated adrenaline diverts blood flow to skeletal muscle, focuses they eye on distant objects and increases blood sugar levels. In addition, stress-activation causes chromaffin cells to release analgesic compounds, termed enkephalins. Together these signaling molecules place the organism into a state for escape or defense. Thus, regulated transmitter release from chromaffin cells plays a critical role in determining the physiological and metabolic status of an organism. It is the overall goal of this laboratory to provide a fundamental understanding of the chromaffin cell as a primary contributor to the 'breed and feed' as well as the 'fight or flight' state. This proposal focuses on the mechanisms utilized by chromaffin cells for transmitter release under basal versus stress activation.Sustained transmitter release requires the recycling of the transmitter-containing secretory granules. Without recycling the cell would soon exhaust its supply. Specialized granule components are retrieved from the cell surface and refilled with transmitter molecules for re-use. It was assumed that this recycling occurred by the same mechanism under all conditions. However, it was recently demonstrated that differences in recycling processes exist under basal versus stress-activation. This proposal outlines a series of experiments designed to test signature events that delineate recycling mechanisms under conditions of basal versus stress activation in mouse adrenal chromaffin cells. It provides experiments that will test the degree of granule fusion and collapse during transmitter release and the degree to which the granule and cell surface components mix, thus defining the complexity of the recycling process required prior to re-generation of viable secretory granules. It will also determine the role of key molecules involved in the recycling mechanism, thus defining points of potential regulation for the re-generation of secretory granules. Techniques employed in these experiments include electrophysiological measures of the surface area of single cells, electrochemical determination of the amount of transmitter released, quantitative fluorescence imaging of cell surface components and molecular disruption of the recycling mechanism. Thus a fundamental description of the mechanisms utilized by adrenal chromaffin cells for the sustained release of adrenaline and enkephalin during the normal 'breed and feed' as well as the 'fight or flight' stress-activated state will be provided.